CN114875100B - Method for improving fermentation yield of natamycin by activating natamycin synthesis in advance - Google Patents
Method for improving fermentation yield of natamycin by activating natamycin synthesis in advance Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
- C12P19/626—Natamycin; Pimaricin; Tennecetin
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
Abstract
The invention relates to a method for improving the fermentation yield of natamycin by activating the synthesis of natamycin in advance. Comprising the following steps: and (3) fermenting the natamycin by adopting a low-nitrogen source fermentation medium, and after fermenting for 64-80 hours, supplementing mother liquor containing high-nitrogen source according to 8-12% of the volume of the medium, and continuing fermenting to obtain the natamycin. According to the invention, the low-nitrogen source fermentation medium is adopted to perform the pre-middle fermentation of the natamycin, and then the high-nitrogen source mother liquor is added into the low-nitrogen source fermentation medium, so that the yield of the natamycin is effectively improved according to the characteristic of the natamycin synthesis stage, and the maximum shake flask fermentation yield is 2.95g/L. After the expansion production is adopted, the yield of the natamycin is 7.88g/L in a 1L fermentation system, which is obviously higher than that of 6.03g/L of the normal fermentation production, and the yield of the natamycin is improved by 20.73 percent.
Description
Technical Field
The invention relates to a method for improving fermentation yield of natamycin by activating natamycin synthesis in advance, and belongs to the technical field of natamycin preparation.
Background
Clinical fungal infection and fungal contamination of food are two major factors threatening human health. Natamycin (also known as pimamycin) is a pharmaceutical productA reducible polyene macrolide antifungal agent of formula C 33 H 47 NO l3 Molecular weight is 665.75 and isoelectric point is 6.5. Natamycin is a polyene chromophore formed by condensation of trehalose with a lactone ring, wherein 4 conjugated double bonds exist in the lactone ring skeleton structure consisting of 26 carbon atoms. Because of the characteristics of stable property, very low oral toxicity, no absorption in intestinal tracts, no anaphylactic reaction, no cross resistance and the like, the compound antibacterial agent not only has better antibacterial effect on the aspects of food raw material preservation and finished product corrosion prevention, but also is widely applied to the aspects of medicines, silage, grain storage, poultry cultivation and the like.
There is a cholesterol oxidase gene (sgnE/pimE/scnE/slnE) in the natamycin biosynthesis gene cluster, and the encoded cholesterol oxidase is not involved in the synthesis and condensation reaction of the mother nucleus and side chain of natamycin. However, recent studies at home and abroad have confirmed that cholesterol oxidase is essential in the biosynthesis of natamycin, and that cholesterol oxidase is involved in the synthesis and accumulation of natamycin (Mendes MV, et al Cholesterol oxidases act as signaling proteins for the biosynthesis of the polyene macrolide pimaricin chemistry and Biology,2007, 14:279-290). Further related studies have shown that addition of finished cholesterol oxidase at the beginning of fermentation can increase the yield of natamycin (Wang M, et al Improvement of natamycin production by cholesterol oxidase overexpression in Streptomyces gilvosporeus, journal of Microbiology and Biotechnology,2016,26 (2): 241-247). Chinese patent literature discloses CN 113481266a method for increasing the fermentation yield of natamycin using cholesterol oxidase, one of the by-products of natamycin fermentation, comprising the steps of cholesterol oxidase preparation and addition. The highest fermentation unit of the natamycin synthesized by the method can reach 6.7g/L. However, the yield of natamycin in the prior art can be further improved.
In addition, natamycin is a secondary metabolite. The fermentation microorganism takes primary metabolite as precursor in a certain growth period, so that the fermentation period of antibiotics such as natamycin has a production delay period for a certain time, and the antibiotics are not synthesized or synthesized at a low rate in the period. How to overcome this problem is therefore a key to further increase the fermentation yield of natamycin.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a method for improving the fermentation yield of natamycin by activating the synthesis of natamycin in advance. The inventor discovers that the culture medium (yeast extract and soybean peptone) with low nitrogen source content in the initial fermentation stage can promote the advanced synthesis of natamycin and shorten the delay period of natamycin; the medium with high nitrogen source content is favorable for accumulation of natamycin in the middle and later stages of fermentation. By utilizing the fermentation characteristics, the concentration of yeast powder and soybean peptone which are mainly sources of nitrogen sources are reduced in the initial stage and the middle-early stage of fermentation, and the high-nitrogen source mother liquor is supplemented in the middle-late stage of fermentation, so that the high-efficiency synthesis of the natamycin is maintained, the yield of the natamycin is improved, and the fermentation cost is reduced.
The technical scheme of the invention is as follows:
a method for increasing fermentation yield of natamycin by activating natamycin synthesis in advance, comprising the steps of:
fermenting the natamycin by adopting a low-nitrogen source fermentation medium, and after fermenting for 64-80 hours, supplementing Gao Danyuan mother liquor according to 8-12% of the volume of the medium, and continuing fermenting to obtain the natamycin;
the low nitrogen source fermentation medium comprises the following components: 1.5 to 2.5g/L of soybean peptone, 0.4 to 0.5g/L of yeast extract, 1.5 to 2.5g/L of sodium chloride, 0.5 to 1.5g/L of crystalline magnesium sulfate and 30 to 50g/L of glucose;
the Gao Danyuan mother liquor is a mixture of yeast extract and soybean peptone, the final concentration of the yeast extract after adding the high nitrogen source mother liquor is 0.5-3 g/L, and the final concentration of the soybean peptone is 2-10 g/L.
According to the invention, the low nitrogen source fermentation medium comprises the following components: soy peptone 2g/L, yeast extract 0.45g/L, sodium chloride 2g/L, crystalline magnesium sulfate 1g/L, glucose 40g/L, pH 7.5.
According to the invention, preferably, the concentration of the soybean peptone in the high nitrogen source mother liquor is 30-50 g/L, and the concentration of the yeast extract is 5-10 g/L; the final concentration of yeast extract after adding high nitrogen source mother liquor was 2.25g/L and the final concentration of soybean peptone was 10g/L.
According to the invention, the strain used for the fermentation production of natamycin is preferably Streptomyces fuscosus (Streptomyces gilvosporeus), streptomyces natakochianus (Streptomyces natalensis), streptomyces knuckaeli (Streptomyces chattanoogensis) or Streptomyces lydicus (Streptomyces lydicus).
According to the invention, the high nitrogen source mother liquor is added after 68 hours of natamycin fermentation production.
According to the invention, preferably, the total time of the natamycin fermentation is 100-150 hours; further preferably 120h.
A method for increasing fermentation yield of natamycin by activating natamycin synthesis in advance, comprising the steps of:
(1) Preparing a low nitrogen source fermentation culture medium and Gao Danyuan mother liquor, and sterilizing for later use;
(2) Under aseptic conditions, coating spores of natamycin producing bacteria in an agar slant culture medium for activation, scraping the activated spores into a seed culture medium, and culturing to obtain seed liquid;
(3) Inoculating the seed solution in the step (2) into a low-nitrogen source fermentation medium according to the inoculation amount of 5%, fermenting and culturing, and after fermenting for 64-80 h, supplementing the mother solution in the step (1) Gao Danyuan into the medium, and continuing fermenting to obtain the natamycin.
The present invention is not limited to the details of the prior art.
The invention has the technical characteristics that:
the nitrogen source is an important component of streptomyces for antibiotic fermentation, natamycin is taken as a reducing antifungal antibiotic produced by streptomyces, the biosynthesis and yield accumulation of the streptomyces take on typical secondary metabolic characteristics, and the fermentation strain grows to a certain period and then starts to be activated or start synthesis, namely a certain period of production delay exists, and the antibiotic is not synthesized or synthesized at a low rate in the period. And the physiological metabolism of antibiotics synthesis, hypha development, cell programmed death (PCD) and the like in streptomyces are closely related to ROS and oxidative stress. Based on the method, seed liquid is transferred into a low-nitrogen source culture medium with relatively poor nutrition for fermentation, so that a great amount of streptomyces ROS is accumulated and an oxidative stress system is activated, thereby activating the synthesis of the natamycin, the ROS can also be used as a reducing substance to resist oxidative damage, further the delay period of the fermentation of the natamycin is shortened, the natamycin is activated and synthesized as soon as possible, and the purposes of improving the yield of the natamycin, shortening the fermentation period and reducing the production cost are achieved.
The invention has the beneficial effects that:
1. according to the invention, the low-nitrogen source fermentation medium is adopted to perform the pre-middle fermentation of the natamycin, and then the high-nitrogen source mother liquor is added into the low-nitrogen source fermentation medium, so that the yield of the natamycin is effectively improved according to the characteristic of the natamycin synthesis stage, and the maximum shake flask fermentation yield is 2.95g/L. After the expansion production is adopted, the yield of the natamycin is 7.88g/L in a 1L fermentation system, which is obviously higher than that of 6.03g/L of the normal fermentation production, and the yield of the natamycin is improved by 20.73 percent.
2. The invention adopts the low nitrogen source fermentation culture medium, and continuously adds the high nitrogen source mother liquor component at the accurate time of the later stage of the culture, thereby achieving the purpose of improving the yield of the natamycin. Compared with the prior fermentation process, the method has the advantages that the synthesis of the natamycin is activated in advance, the yield is improved, the delay period of the synthesis of the natamycin is effectively shortened by 24 hours, the production cost is reduced, and great economic benefit is generated.
Drawings
FIG. 1 shows the effect of low nitrogen source fermentation medium and normal fermentation medium on natamycin synthesis.
FIG. 2 shows the effect of addition of different concentrations of high nitrogen source mother liquor on natamycin synthesis.
FIG. 3 shows the effect of different addition times of high nitrogen source mother liquor on natamycin synthesis.
Detailed Description
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
The materials involved in the invention are all common commercial products.
Preparing a seed solid culture medium: 10g/L of glucose, 3g/L of malt extract powder, 3g/L of yeast extract, 5g/L of peptone, 15g/L of agar powder, pH7.0, sterilizing at 121 ℃ for 20min, and cooling for later use.
Preparing a shake flask seed culture medium: 20g/L glucose, 5g/L yeast extract, 40g/L soybean cake powder, pH 7.2, sterilizing at 121deg.C for 20min, and cooling.
Preparation of a fermentation tank natamycin liquid seed culture medium: glucose 10g/L, yeast extract 3g/L, soybean peptone 5g/L, malt extract 3g/L; 1.40g/L of defoamer; 7.0 to 7.2, sterilizing at 121 ℃ for 20min, and cooling for later use.
Preparation of low nitrogen source (LS) fermentation medium: soy peptone 2.0g/L, yeast extract 0.45g/L, sodium chloride 2g/L, crystalline magnesium sulfate 1g/L, glucose 60g/L, pH7.5; sterilizing at 121deg.C for 20min, and cooling.
Preparation of normal (HS) fermentation medium: 20g/L of soybean peptone, 4.5g/L of yeast extract, 2g/L of sodium chloride, 1g/L of crystalline magnesium sulfate, 60g/L of glucose and pH7.5; sterilizing at 121deg.C for 20min, and cooling.
Preparing Gao Danyuan mother liquor: the soybean peptone and the yeast extract are respectively prepared into mother solutions of 10+2.25g/L, 20+4.5g/L, 50+11.25g/L, 100+22.5g/L and 200+45g/L, sterilized at 121 ℃ for 20min, and cooled for later use.
Determination of pH: direct measurement was performed using a mertler pH meter.
Example 1
A method for increasing fermentation yield of natamycin by activating natamycin synthesis in advance, comprising the steps of:
(1) Under the aseptic condition, activating the streptomyces fuscosus on a seed solid culture medium, scraping spores from an agar slant culture medium after the activation, inoculating the spores into a shake flask seed culture medium, and carrying out shake culture for 24 hours in a shaking table at the temperature of 29 ℃ at the rotating speed of 220rpm to obtain seed liquid;
(2) Transferring the seed liquid into a low nitrogen source fermentation medium (LS) and a normal fermentation medium (HS) respectively according to the inoculation amount of 5%, wherein the fermentation medium is contained in 500mL triangular shake flasks, the volume of each 500mL triangular shake flask-packed fermentation medium is 50mL, each experimental group is 6 bottles, 220rpm, and fermentation is completed at 29 ℃ for 144 hours, so as to obtain the natamycin fermentation liquid.
The natamycin content of the above group 2 was measured every 24 hours and the results are shown in figure 1. The natamycin content determination method is carried out according to the method specified in national standard GB 25532-2010.
As can be seen from the results of fig. 1, the yield of natamycin in the LS low nitrogen source fermentation medium group was significantly higher than that in the HS normal fermentation medium group in the pre-fermentation period (< 48 h) and mid-pre-fermentation period (48-72 h); and in the middle period (> 72 h-120 h) and the later period (> 120 h) after synthesis of the natamycin, the natamycin yield of the HS normal fermentation medium group is obviously higher than that of the LS low nitrogen source fermentation medium group. The low nitrogen source content in the early stage is beneficial to the activation of the synthesis of the natamycin, and the nitrogen source sterol in the middle and later stages has a key effect on the synthesis of the natamycin. According to the data result, the biosynthesis of the natamycin is activated in advance in the low-nitrogen source fermentation medium, so that the reduction of the nitrogen source content in the middle-before-natamycin culture process can effectively improve the middle-before-natamycin yield and further improve the overall yield of the natamycin.
Example 2
A method for increasing fermentation yield of natamycin by activating natamycin synthesis in advance, comprising the steps of:
(1) Under the aseptic condition, activating the streptomyces fuscosus on a seed solid culture medium, scraping spores from an agar slant culture medium after the activation, inoculating the spores into a shake flask seed culture medium, and carrying out shake culture for 24 hours in a shaking table at the temperature of 29 ℃ at the rotating speed of 220rpm to obtain seed liquid;
(2) Transferring the seed liquid into a low nitrogen source fermentation medium (LS) according to the inoculation amount of 5%, wherein the fermentation medium is contained in 500mL triangular shake flasks, the volume of each 500mL triangular shake flask is 50mL, each experimental group is 6 bottles, 220rpm and 29 ℃ are used for fermentation culture, high nitrogen source mother liquor is added during the fermentation culture for 72 hours, the fermentation is continued for 144 hours, and the fermentation is finished to obtain natamycin fermentation liquor; wherein the added amount of the mother liquor of the high nitrogen source is 10% (volume ratio) to the culture medium, and the final concentrations of the soybean peptone and the yeast extract are respectively 0g/L (equal volume of sterilized water, control group), 1.0+0.225g/L (experimental group 1), 2.0+0.45g/L (experimental group 2), 5.0+1.125g/L (experimental group 3), 10+2.25g/L (experimental group 4) and 20+4.5g/L (experimental group 5) of the fermentation culture, and are shown in Table 1.
The natamycin content of the above 6 groups was measured and the results are shown in figure 2. The natamycin content determination method is carried out according to the method specified in national standard GB 25532-2010.
TABLE 1 high Nitrogen Source mother liquor addition concentration
As can be seen from the results of fig. 2, the yield of natamycin is significantly improved in experimental group 4 compared with the control group, and the yield of natamycin reaches the highest value and the improvement amplitude reaches 46.6% at 120h. After the high nitrogen source mother liquor is added, the test group 2, the test group 3 and the test group 5 are improved to different degrees, but the improvement amount is not obvious. According to the above data, the final concentration of the high nitrogen source mother liquor added in the examples of the present invention was 10g/L soybean peptone+2.25 g/L yeast extract, which is the final concentration after the addition.
Example 3
A method for increasing fermentation yield of natamycin by activating natamycin synthesis in advance, comprising the steps of:
(1) Under the aseptic condition, activating the streptomyces fuscosus on a seed solid culture medium, scraping spores from an agar slant culture medium after the activation, inoculating the spores into a shake flask seed culture medium, and carrying out shake culture for 24 hours in a shaking table at the temperature of 29 ℃ at the rotating speed of 220rpm to obtain seed liquid;
(2) Transferring the seed solution into a low nitrogen source fermentation medium (LS) according to an inoculum size of 5%, wherein the fermentation medium is contained in 500mL triangular shake flasks, the volume of each 500mL triangular shake flask is 50mL, each experimental group is 6 bottles, 220rpm,29 ℃ is used for fermentation culture, high nitrogen source mother liquor (soybean peptone with a final concentration of 10g/L and 2.25g/L yeast extract) is added in the fermentation culture process, the addition amount is 10% of the volume of the fermentation medium, the fermentation is continued, the total fermentation time is 144h, and the natamycin fermentation liquor is obtained after the fermentation is finished; the adding time of the high nitrogen source mother liquor is 64 hours (experiment group 1), 68 hours (experiment group 2), 72 hours (experiment group 3), 76 hours (experiment group 4) and 80 hours (experiment group 5) after fermentation culture, and the high nitrogen source mother liquor is not added as a control group, and the details are shown in Table 2.
The natamycin content of the above 6 groups was measured and the results are shown in fig. 3. The natamycin content determination method is carried out according to the method specified in national standard GB 25532-2010.
TABLE 2 different addition times of high Nitrogen Source mother liquor
As shown in FIG. 3, the experimental groups 1 to 5 have improved natamycin yield of 120 hours, which is 3.2 to 60.72 percent compared with the control group. However, adding the high nitrogen source mother liquor at different times has an effect on the yield of natamycin, the yield of natamycin in the experimental group 1 is improved by 42.52% compared with that in the control group, the yield of natamycin in the experimental group 2 is improved by 60.72% compared with that in the control group, the yield of natamycin in the experimental group 4 and the yield of natamycin in the experimental group 5 are reduced compared with that in the experimental group 2, but the yield of natamycin in the experimental group is still improved by more than 3.2% compared with that in the control group.
Example 4
A method for increasing fermentation yield of natamycin by activating natamycin synthesis in advance, comprising the steps of:
(1) Under the aseptic condition, activating the streptomyces fuscosus on a seed solid culture medium, scraping spores from an agar slant culture medium after the activation, inoculating the spores into a shake flask seed culture medium, and carrying out shake culture for 24 hours in a shaking table at the temperature of 29 ℃ at the rotating speed of 220rpm to obtain seed liquid;
2) Inoculating the seed solution into a low nitrogen source fermentation medium (LS) according to an inoculum size of 10%, wherein the volume of the fermentation medium before inoculation is 0.8L, filling the fermentation medium into a 1L full-automatic fermentation tank, fermenting at 29 ℃, controlling the dissolved oxygen DO value in the fermentation process to be between 30 and 40%, and beginning to flow 20% concentration NaOH when the pH value in the initial stage of culture naturally drops to 6.00+/-0.10 from the initial pH value and maintaining the pH value to be between 6.00+/-0.20 until the fermentation is finished; adding 40% glucose solution by mass percent in the fermentation process, controlling the glucose content to 15.00+/-5.00 g/L all the time, adding high nitrogen source mother liquor (100 g/L soytone+22.5 g/L yeast extract) after 68h of fermentation, wherein the addition amount is 10% of the volume of a fermentation medium, the final concentration of soytone in a post-fermentation system is 10g/L, the final concentration of yeast extract is 2.25g/L, and continuously fermenting for 120h to obtain natamycin; a control group was set, to which an equal volume of sterile water was added.
The natamycin content of the above group 2 was measured and the results are shown in table 3. The natamycin content determination method is carried out according to the method specified in national standard GB 25532-2010.
TABLE 3 Natamycin fermentation in 1L fermentation System
As can be seen from Table 3, the yield of natamycin in the experimental group is 7.78g/L, which is significantly higher than that of the natamycin in the control group without adding the mother liquor with high nitrogen source, which is 10% of the volume after fermentation for 68 hours, and the yield is improved by 24.88%. The verification result of a 1L fermentation system shows that the addition of 10% by volume of high-nitrogen source mother liquor can significantly improve the yield of natamycin.
Claims (6)
1. A method for increasing the fermentation yield of natamycin by activating the natamycin synthesis in advance, comprising the steps of:
fermenting the natamycin by adopting a low-nitrogen source fermentation medium, and after fermenting for 64-80 hours, supplementing Gao Danyuan mother liquor according to 8-12% of the volume of the medium, and continuing fermenting to obtain the natamycin;
the low nitrogen source fermentation medium comprises the following components: soy peptone 2g/L, yeast extract 0.45g/L, sodium chloride 2g/L, crystalline magnesium sulfate 1g/L, glucose 60g/L, pH7.5;
the Gao Danyuan mother liquor is a mixture of yeast extract and soybean peptone, the final concentration of the yeast extract after adding the high nitrogen source mother liquor is 2.25g/L, and the final concentration of the soybean peptone is 10g/L;
the strain used for fermenting and producing the natamycin is Streptomyces fuscosus @ or Streptomyces fuscosus @Streptomyces gilvosporeus）。
2. The method for improving the fermentation yield of natamycin according to claim 1, wherein the concentration of soybean peptone in the high nitrogen source mother liquor is 30-50 g/L, and the concentration of yeast extract is 5-10 g/L.
3. The method for improving the fermentation yield of natamycin according to claim 1, wherein the mother liquor of the high nitrogen source is added after 68 hours of the fermentation production of natamycin.
4. The method for improving the fermentation yield of natamycin according to claim 1, wherein the total fermentation time of natamycin is 100-150 h.
5. The method for increasing fermentation yield of natamycin according to claim 1, wherein the total time of the natamycin fermentation is 120 hours.
6. The method for improving the fermentation yield of natamycin according to claim 1, characterized in that the method comprises the following steps:
(1) Preparing a low nitrogen source fermentation culture medium and Gao Danyuan mother liquor, and sterilizing for later use;
(2) Under aseptic conditions, coating spores of natamycin producing bacteria in an agar slant culture medium for activation, scraping the activated spores into a seed culture medium, and culturing to obtain seed liquid;
(3) And (3) inoculating the seed liquid in the step (2) into a low-nitrogen source fermentation medium according to an inoculum size of 5%, fermenting and culturing, and after fermenting for 64-80 h, supplementing the mother liquid in the step (1) Gao Danyuan into the medium, and continuing fermenting to obtain the natamycin.
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